Polymerase chain reaction amplification of long DNA targets: application to analysis of chromosomal translocations in human B-cell tumors (review).
Chromosomal translocations in human leukemias generate fusion transcripts containing messages from two genes involved in the translocation, and these have been the targets for reverse transcriptase-mediated polymerase chain reaction (PCR). In contrast, many of the translocations in B-cell tumors involve immunoglobulin gene (IG) loci, and coding regions of the oncogenes on partner chromosomes are not interrupted by the translocation. Therefore, targets for PCR amplification are single-copy oncogene/IG fusion sequences within the complex genomic DNA. We present here a novel strategy for detection of translocations in B-cell tumors on the basis of long-distance (LD-) PCR that is capable of amplifying up to 30 kb of DNA. LD-PCR is a general method using primer pairs designed for distinctive regions of IG and oncogenes involved in translocations, and amplifying long DNA fragments encompassing the oncogene/IG junction. LD-PCR is capable of detecting virtually all the important translocations in B-cell tumors, including t(8;14)(q24;q32), t(14;18)(q32;q21), t(3;14)(q27;q32) and its variants. We show here that LD-PCR can substitute for time-consuming Southern blot hybridization in the rapid detection of these translocations. Furthermore, as amplified fragments obtained by LD-PCR contained exons and flanking sequences of the oncogenes and IGs, restriction analysis and nucleotide sequencing of the products refined the characteristics of translocations.
Akasaka
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First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54-Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-01, Japan.
Akasaka
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Ohno
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D002874:Chromosome Mapping; D002877:Chromosomes, Human; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000235:genetics; D005091:Exons; D005784:Gene Amplification; D006801:Humans; D015448:Leukemia, B-Cell / Q000235:genetics*; D016393:Lymphoma, B-Cell / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics*
D017931:DNA Primers; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors